Mutations in Zinc Finger in the Cerebellum (ZIC) genes and the Glioma-associated Oncogene Homolog (GLI) have been linked to multiple debilitating, congenital brain disorders in humans. In particular, abnormalities in these proteins play a critical role in patterning disorders such as the Dandy-Walker Malformation, holoprosencephaly, and Greig Cephalopolysyndactyly Syndrome, with ramifications both for intellectual capacity and life expectancy. The implication of these genes in disease states has been proven in a variety of ways in the literature. For example, viable mouse models for Dandy-Walker are created through a mutation that fuses ZIC1and ZIC4, deleting zinc-finger domains from each. The precise mutations that cause these phenotypes in humans, however, have remained elusive. Attempts to examine Dandy-Walker and other patients have failed to uncover de novo mutations in ZIC and GLI proteins in a simple, causal relationship to the disease state. In other cases, substantial heterogenity exists between observed phenotypes even when mutations that cause a given disorder are present. We theorize that both of these issues arise from an incomplete understanding of ZIC functionality and interaction patterns. Until recently, the canonical model of zinc-finger proteins such as ZIC and GLI was that individual fingers in the protein never interacted with one another, but only with DNA. Recent research, however, has established that ZIC and GLI proteins likely do not function according to canonical models. Instead, they are members of a newly identified class of Cys2His2 proteins called two-finger or interacting-finger proteins, a subset characterized by interactions between fingers in a single protein. Furthermore, it has recently been proven that ZIC and GLI interact not only internally, from finger to finger, but with each other, in protein- protein interactions that remain understudied. These new observations, taken collectively, lead us to hypothesize that the disruption of ZIC-ZIC and ZIC-GLI interactions by mutations, and not single or paired mutations alone, are causal in the patterning disorders associated with these genes, particularly in Dandy-Walker Syndrome. We therefore propose a two-year study in which we will test whether interactions exist between ZIC proteins via yeast two-hybrid screens, confirm ZIC-ZIC and ZIC-GLI interactions via Isothermal Titration Calorimetry (ITC), and determine if mutations associated with disease states disrupt interactions between ZIC-ZIC and ZIC- GLI. Our hope is that the work will lead not only to an understanding of the molecular mechanisms underlying disorders associated with ZIC and GLI genes, but also to better prognostic and diagnostic clinical tests that will create opportunities for early intervention and lead to healthier and longer lives for children with some of these disorders. PUBLIC HEALTH RELEVANCE: This project will determine the role protein-protein interactions play in a set of devastating congenital brain disorders that manifest during human development. Guided by our recent findings on a class of zinc-finger proteins with a unique interaction domain, we will examine how mutation in these proteins causes or contributes to disease states, laying the foundation for future prognostic and diagnostic tools that will permit early intervention in developmental patterning disorders such as the Dandy-Walker Malformation, holoprosenchephaly, Grieg Cephalopolysyndactyly Syndrome, and others. [unreadable] [unreadable] [unreadable]